SEROVAR PROFILE AND DETECTION OF INVA VIRULENCE GENE AMONG NON-TYPHOIDAL SALMONELLAE SEROVARS ISOLATED FROM ACUTE GASTROENTERITIS CASES IN COASTAL KARNATAKA, SOUTHERN INDIA

  Objective: Non-typhoidal salmonellosis is one of the leading zoonosis in the world caused by non-typhoidal Salmonella (NTS). Invasive infections with NTS serovars occurs due to the presence of virulence genes like invA along with the immunosuppressive conditions of the patient. The study was conducted to isolate and identify the NTS serovars and their antimicrobial resistance profile from patients with diarrhea and also to detect the virulence marker – invA gene among these NTS serovars.Methods: A prospective cross-sectional study was conducted from January 2015 to December 2016 at the Enteric Diseases Division, Kasturba Medical College, Manipal. 1218 fecal specimens were collected from patients with diarrhea and before antibiotic treatment. NTS serovars were identified, serotyped and then screened for the presence of invA virulence gene.Results: A total of 33 (2.7%) NTS was isolated. Salmonella typhimurium (33.34%) was predominant followed by Salmonella oslo (30.3%). Out of 33 NTS, invA was positive for 28 isolates (84.8%) of which 25 (89.3%) patients were febrile which was statistically significant (p=0.000).Conclusion: Non-typhoidal salmonellosis is an emerging global infection among immunocompromised patients. Our study showed an association between the invA gene and febrile illness among the patients suffering. Thus, this study highlights the importance of invA as a significant marker for bloodstream invasion

letter to tHe eDitor

Bhat M, Lu Y, Marcil V, et al. Tumour necrosis factor-alpha polymorphism increases the risk for nonvariceal upper gastrointestinal bleeding in patients taking proton pump inhibitors. Can J Gastroenterol Hepatol 2014;28(9):488. To the Editor: Nonvariceal upper gastrointestinal bleeding (NVUGIB) is associated with significant morbidity, affecting 50 to 150 per 100,000 adults annually (1). Patients with NVUGIB may present with melena, hematochezia or coffee-ground emesis, often accompanied by a decrease in hemoglobin levels and even hemodynamic instability. Nonsteroidal anti-inflammatory drugs (NSAIDs) and Helicobacter pylori infection are the principal risk factors for NVUGIB, accounting for >95% of cases. Taking proton pump inhibitors (PPIs) is a known protective factor against NVUGIB. There has recently been growing interest in a possible genetic predisposition to NVUGIB, with investigation of single nucleotide polymorphisms (SNPs) associated with bleeding events. Of particular interest have been mutations in proinflammatory genes or genes that regulate NSAID/PPI metabolism, which may contribute to excessive inflammation and ulceration in the context of H pylori infection and NSAID use. A recent pharmacogenomic study The above literature describing a genetic predisposition for bleeding events applies principally to East Asian patient populations. Therefore, we decided to study whether such genetic associations could be elicited in the Canadian context. We performed a pilot study to assess the association of SNPs involved in NSAID metabolism (CYP2C9) and inflammatory response (TNF-α) with NVUGIB events. Patients who were part of the REASON-II NVUGIB study population at the McGill University Health Centre (Montreal, Quebec) were recruited (5). Study controls were asymptomatic patients undergoing screening colonoscopy, and excluded if there was any history of NVUGIB. DNA extracted from serum was genotyped for SNPs in the proinflammatory TNF-α (rs1799724, rs1800630, rs1799964) and NSAID-metabolizing CYP2C9 genes (rs1799853, rs1057910). Using STATA version 10, we assessed for any association between SNPs and NVUGIB events using logistic regression analysis and stratifying according to H pylori status, NSAID and PPI use. Our study included 23 patients and 46 controls of comparable age and sex, with NSAID (26.1% versus 6.7%) and PPI use (21.7% versus 13.0%) being more prevalent among patients. The TNFα1031C SNP, a proinflammatory cytokine polymorphism, was more common among patients with NVUGIB (OR 2.2 [95% CI 0.9 to 5.1]; P=0.084), particularly among those using PPIs (OR 20.0 [95% CI 0.9 to 429.9]; P=0.056) or not taking NSAIDs (OR 3.2 [95% CI 1.1 to 9.0]; P=0.027) at the time of the bleeding event. There was a trend in association of the TNF-α863A SNP with NVUGIB in patients not taking NSAIDs (OR 2.7 [95% CI 0.9 to 8.6]; P=0.071). We did not detect an association between CYP2C9 polymorphisms and NVUGIB, a result similar to that obtained in the study by Musumba et al (2). In conclusion, our pilot study demonstrates that TNF-α1031C SNP confers a risk for NVUGIB events among patients taking PPIs, a finding compatible with previous studies showing increased risk for peptic ulceration with this particular SNP (3

The impact of preexisting and post-transplant diabetes mellitus on outcomes following liver transplantation

Background: Diabetes mellitus (DM) is said to adversely affect transplant outcomes. The aim of this study was to investigate the impact of pre-existing and post-transplant DM on liver transplant (LT) recipients.Method: A single centre retrospective analysis of prospectively collected data of LT recipients (1990–2015) was undertaken.Results: Of the 2,209 patients, 13% (n=298) had Pre-DM, 16% (n=362) developed PTDM, 5% (n=118) developed transient hyperglycemia (t-HG) post-LT, and 65% (n=1,431) never developed DM (no DM). Baseline clinical characteristics of patients with PTDM was similar to that of patients with pre-DM. Incidence of PTDM peaked during first-year (87%) and plateaued thereafter. On multivariate analysis (Bonferroni-corrected), non-alcoholic fatty liver disease and the use of Tacrolimus and Sirolimus use were independently associated with PTDM development. Both Pre-DM and PTDM patients had satisfactory and comparable glycaemic control throughout the follow-up period. Those who developed t-HG seems to have a unique characteristic compared to others. Overall, 9%, 5%, and 8% developed end-stage renal disease (ESRD), major cardiovascular event (mCVE), and de novo cancer, respectively. Both Pre-DM and PTDM did not adversely affect patient survival, re-LT, or de novo cancer. The risks of ESRD and mCVE were significantly higher in patients with Pre-DM followed by PTDM and no DM.Conclusions: In this largest non-registry study, patients with pre-DM and PTDM share similar baseline clinical characteristics. Pre-DM increases the risk of ESRD and mCVE; however, patient survival was comparable to those with PTDM and without diabetes. Understanding the impact of PTDM would need prolonged follow-up

Characteristics of liver transplant candidates delisted following recompensation and predictors of such delisting in alcohol-related liver disease: a case-control study

Whether and when recovery beyond the need for transplant may occur in patients listed for decompensation remains unclear. This study aimed to investigate the characteristics of patients delisted following recompensation. Seventy-seven patients who were listed between 2005 and 2015 for decompensation, but later delisted following recompensation were included. Alcohol-related liver disease (ALD) was the underlying etiology in the majority (n=47, 61%). Listing characteristics of these patients were compared with those of decompensated ALD patients who either underwent deceased donor liver transplantation or died on the waiting list. The model for end-stage liver disease (MELD) score <20 and serum albumin ≥32g/l at listing were the only independent predictors of recompensation/delisting in ALD. The probability of recompensation was 70% when both factors were present at listing. Interestingly, about a tenth of decompensated ALD patients who died on the waiting list (median duration on waiting list 11 months) and a quarter of decompensated ALD patients who underwent living donor liver transplantation (median duration on waiting list 2 months) also had both factors at listing. In conclusion, ALD seems to be the most favorable etiology for recompensation beyond the need for transplantation. Both MELD and serum albumin at listing independently predict recompensation/delisting in ALD. It seems advisable to implement a period of observation for ALD patients with both favorable factors, before embarking on living donor liver transplantation

Liver transplantation is a preferable alternative to palliative therapy for selected patients with advanced hepatocellular carcinoma

Background: Patients with hepatocellular carcinoma (HCC) beyond the traditional criteria (advanced HCC) are typically offered palliation, which is associated with a 3-year survival rate lower than 30%. This study aimed to describe the outcomes for a subset of patients with advanced HCC who satisfied the Extended Toronto Criteria (ETC) and were listed for liver transplantation (LT). Materials & Methods: All patients listed in the Toronto liver transplant program with HCC beyond both the Milan and University of California, San Francisco criteria were included in this study. Data were extracted from the prospectively collected electronic database. All radiological images were reviewed by two independent radiologists. The primary endpoint was patient survival. Results: Between January 1999 and August 2014, 96 patients with advanced HCC were listed for LT, and 62 (65%) of these patients received bridging therapy while on the waiting list. Bridging therapy led to a significant reduction in tumor progression (p=0.02) and tumor burden (p <0.001). The majority of those listed underwent LT (n=69, 72%). Both tumor progression on waiting list (HR 4.973 [1.599 – 15.464], p=0.006) and peak AFP ≥400ng/ml (HR 4.604 [1.660 – 12.768], p=0.003) were independently associated with waiting list dropout. Post-LT HCC recurrence occurred in 35% (n=24). Among those with HCC recurrence, survival was significantly better for those who received curative treatment (p=0.004). The overall actuarial survival rates from the listing were 76% at 1 year, 56% at 3 years, and 47% at 5 years, and the corresponding rates from LT were 93%, 71%, and 66%. Conclusion: LT provides significantly better survival rates than palliation for patients with selected advanced HCC

A Network-based Analysis of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a high-fatality cancer with complex pathogenesis, often arising in the setting of various chronic liver diseases when it is too late to offer any curative therapy. This PhD thesis is divided into two parts, the first of which was completed at McGill University under the supervision of Dr. Nahum Sonenberg, and evaluated inhibition of translation downstream of mTOR. This work was performed in the context of much interest in inhibiting the mTOR pathway in HCC, given its identification as a dominant pathway in this cancer. In the first part of this thesis, I evaluated how metformin inhibits HCC growth, by decreasing translation of anti-apoptotic proteins such as Mcl-1 downstream of mTOR. In the second part of this thesis, I sought to better understand the role of the mTOR pathway in relation to other commonly dysregulated pathways in HCC. All publicly available, high-throughput gene expression data in HCC was curated at http://ophid.utoronto.ca/CDIPLiver. Clinical variables such as etiology of liver disease and liver function parameters, important to understanding clinical relevance of gene expression data, were only available in around 50% of studies. Betweenness centrality calculation was performed on genes differentially expressed between HCC and corresponding background liver tissue, and revealed Estrogen Receptor 1 (ESR1) to be most central to the HCC Protein-Protein Interaction (PPI) network. HCC cells transfected with ESR1 and exposed to estradiol had significantly decreased proliferation and viability. Gene expression revealed that ESR1 upregulation significantly affected cellular processes such as histone methylation, transcription, and cell cycle. High expression of ESR1 in HCC tumors was found to be protective. Finally, we curated all publicly available whole exome sequencing, DNA methylation, microRNA, and proteomics data in HCC. Pathway analysis was performed on significantly dysregulated genes, and overlapping pathways identified. The following pathways overlapped among the 5 different datasets: Epidermal Growth Factor (EGFR), B1-integrin and axon guidance proteins, suggesting pathway dependencies in HCC. A systems biology approach to HCC allowed us to elucidate the role of sex-specific differences and importance of specific pathways in HCC biology, providing biological rationale for a more targeted therapeutic approach to HCC.Ph.D

Expression of PCSK9 in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is an often fatal condition due to late diagnosis, resistance to existing anticancer agents, as well as underlying liver disease that can limit the use of hepatotoxic chemotherapy. Proprotein convertases (PCs) are serine proteases that convert a variety of growth factors, cell surface glycoproteins, receptors and metalloproteinases into their active forms, thus regulating the biological activity of these proteins. PCs have been found to be upregulated in various malignancies. Growth factors implicated in HCC, such as IGF-1, HGF, VEGF and PDGF, have all been shown to be converted into their active forms by PCs. In this study, I explored the hypothesis that expression of proprotein convertases, specifically PCSK9, furin and PC5, is elevated in HCC. This was evaluated through construction of a Tissue Microarray and staining for these proteins. We found that PCSK9 expression was significantly downregulated in HCC tumours associated with poorer survival. PCSK9 is upregulated in the context of liver regeneration and has been involved in cholesterol metabolism, with development of monoclonal antibodies against PCSK9 to treat hypercholesterolemia. Its altered expression in aggressive HCC tumours potentially indicates that HCC is able to modulate its local microenvironment to enable a constant energy supply. There has recently been a move in oncology research to study suppression of suppress tumour growth by modifying energy supply and metabolism (for eg, metformin in prostate and breast cancer). Further confirmation at the mRNA level is required to confirm the altered expression of PCSK9, however this appears to be a promising finding and potential chemotherapeutic target.Contexte et hypothèses: Le carcinome hépatocellulaire (CHC) est le 5e cancer le plus courant dans le monde entier et la 3ème cause de décès par cancer dans le monde entier, avec une survie médiane à 5 ans de 8,9%. La reconnaissance tardive en raison du manque de biomarqueurs pour détecter la maladie résécable, une résistance aux agents anticancéreux, ainsi qu'une maladie du foie sous-jacente limitant l'utilisation de chimiothérapie hépatotoxique sont des facteurs qui diminuent le taux de survie. Les proprotéines convertases (PCs) sont des sérine-protéases qui convertissent une variété de facteurs de croissance, glycoprotéines de surface cellulaire, les récepteurs, et les métalloprotéinases à leurs formes actives, contrôlant ainsi l'activité biologique de ces protéines. On a démontré l'expression augmentée de PCs dans de diverses tumeurs malignes. On a prouvé que les facteurs de croissance impliqués dans le CHC, tels que l'IGF-1, HGF, VEGF et PDGF, sont convertis à leurs forme actives par les PC. Notre hypothèse est que l'expression de proprotéines convertases est élevée dans le CHC, permettant l'activation de différentes protéines essentielles dans le développement et la progression du CHC. L'objectif de recherche était d'évaluer l'expression des PCs PCSK9, furine et PC5 dans le CHC par rapport aux stroma environnant, zones péri-cirrhotiques, et foie normal afin de déterminer si un gradient d'expression existe. PCSK9 en particulier est connu comme étant plus exprimé chez le foie régénérateur post-hepatectomie. Les diapositives de pathologie de CHC stockés dans le département de pathologie du CUSM ont été examinés par une pathologiste, et les zones appropriées (tumeur de CHC, interface de tumeur et du foie, le foie cirrhotique, et d'autres échantillons d'hépatite et de foie normal) dans les blocs de tissu correspondants ont été creusés et ont été incorporées dans un microarray de tissu (TMA). Des lignes cellulaires de CHC etablies, dont le HepG2 et le Huh7, avec des profils d'expression de PC connus, ont été incorporées sous forme de pastilles de cellules dans la TMA, afin de servir de témoins positifs et négatifs. La TMA a été sectionnée en diapositives, qui ont été colorées avec des anticorps de la PCSK9, furine et PC5. On a découvert que le niveau d'expression de PCSK9 était diminuée dans les CHC avec un pire prognostique. L'expression augmentée de PCSK9 dans les CHC plus aggressifs pourrait indiquer un rôle du PCSK9 dans la tumorigenèse, directement ou indirectement. Il se peut que les CHCs plus aggressifs sont capables de modifier l'environnement local pour apprivoiser l'énergie métabolique, et que le PCSK9 permet que le cholestérol soit utilisé comme source d'énergie. La confirmation de son importance fonctionnelle avec mRNA pourrait potentiellement mener au développement de chimiothérapie ciblée avec des anticorps contre le PCSK9 (stratégie en étude pour l'hypercholestérolémie). Compte tenu des options chimiothérapeutiques actuellement limitées pour le CHC, une telle constatation pourrait améliorer la prise en charge clinique du CHC